Anti hog1 antibody yc20

Frozen cell pellets were resuspended in 250 μl of 1× SDS loading buffer. The cell suspensions were vigorously ground using glass beads at 4°C and centrifuged at 9,200 g for 10 min at 4°C. The supernatants (cell lysates) were either immediately used, or frozen in liquid nitrogen and stored at −80°C. Aliquots (12 μl) of the cell lysates were mixed with 10 μl of 1× SDS loading buffer, boiled for 5 min, and separated by Bis‐tris SDS–PAGE containing 40 μM Phos‐tag (Phos‐tag SDS–PAGE). After electrophoresis, the bound phosphopeptides were released from Phos‐tag acrylamide by soaking the gel first in a solution consisting of 25 mM Tris, 192 mM glycine, 20% (v/v) methanol, and 1.0 mM EDTA for 15 min twice, and then in a solution consisting of 25 mM Tris, 192 mM glycine, and 20% (v/v) methanol for 20 min. The proteins in the gel were transferred onto Amersham™ Hybond ™ P 0.45 μm PVDF membranes (GE Healthcare). Hog1 was detected by immunoblotting using the anti‐Hog1 antibody yC20 (Santa Cruz Biotechnology). The slow‐ and fast‐migrating Hog1 bands represent phosphorylated and unphosphorylated forms, respectively. Enhanced chemiluminescence images were digitally captured using the ChemiDoc XRS Plus (Bio‐Rad) equipped with a charge‐coupled‐device camera. Quantitation of band intensity was carried out using the Image Lab program (version 4.1, Bio‐Rad).

After electrophoresis (SDS–PAGE, or Phos‐tag SDS–PAGE), proteins were transferred onto Amersham™ Protran™ 0.45 μm nitrocellulose membranes (GE Healthcare) or onto Amersham™ Hybond ™ P 0.45 μm PVDF membranes (GE Healthcare). Phosphorylated Hog1 was detected by immunoblotting using the anti‐phospho‐p38 MAPK (T180/Y182) antibody #9211 (Cell Signaling Technology). Hog1 was detected using the anti‐Hog1 antibody yC‐20 (Santa Cruz Biotechnology). FLAG‐Hog1 was detected by anti‐FLAG M2 antibody (Sigma‐Aldrich). Pbs2‐HA was detected by anti‐HA antibody F‐7 (Santa Cruz Biotechnology). Phosphorylation of Pbs2 Thr‐518 was detected by polyclonal anti‐Pbs2 phospho‐T518 antibody custom produced by SCRUM Inc. Phosphorylated Kss1 and Fus3 were detected using the anti‐phospho‐p44/42 MAPK (Erk1/2; T202/Y204) Rabbit monoclonal antibody #4370 (Cell Signaling Technology).